Findings from the CAPRISA 004 Phase IIb clinical prevention trial in women who applied the HIV-1 nucleotide reverse transcriptase inhibitor (NRTI) Tenofovir (TFV) before and after sexual intercourse, illustrate the potential for microbicides as relevant strategies to prevent mucosal transmission of HIV-1. TFV reduced HIV-1 infection by an estimated 39% overall, and by 54% in women with a compliance rate of 80%. The reason(s) underlying the limited efficacy of TFV in this clinical trial are unknown but may reflect the lack of understanding of the interplay between co-factors that enhance mucosal transmission of HIV-1 and lead microbicides. Sexually transmitted pathogens (STP) highly prevalent in HIV-endemic populations enhance HIV-1 infection and transmission. It is well established that STP activate Toll Like Receptors (TLR), a family of pathogen recognition receptors to elicit a defense immune response, which includes cell activation and differentiation, secretion of type I interferon's and antimicrobial peptides, ad secretion of pro-inflammatory cytokines and chemokines that recruit and activate HIV-1 target cells. Thus, by activating TLR signaling pathways, STP may enhance pro-inflammatory innate immune responses that if dominant may override protective antiviral responses and decrease the anti-HIV-1 activity of microbicide candidates. As immune cell activation by Herpes Simplex Virus Type-2 (HSV-2) is associated with enhanced HIV-1 infection and replication, our proposal will test the hypothesis that TLR activation by STP modulates pro- inflammatory and antiviral innate immune responses to enhance HIV-1 infection, thereby compromising the efficacy of HIV-1 microbicides. Bacterial, fungal and viral pathogens highly prevalent in HIV-1-infected women bind to, and activate specific TLR. Thus, it is highly likely we will see a differential response according to the TLR signaling pathway activated by STP. Specific Aim 1 will define whether TLR activation by their cognate ligands modulates the anti-HIV-1 activity of lead microbicides by impacting innate pro-inflammatory and antiviral immune responses in the cervicovaginal mucosa. We will focus on TFV, the first NRTI vaginal formulation with demonstrated clinical effectiveness against HIV-1 infection, UC781 a non-nucleotide reverse transcriptase inhibitor with demonstrated clinical safety after vaginal application, and elvitegravr an integrase inhibitor with post-exposure profilaxis properties. Specific Aim 2 will explore TLR-dependent immunological mechanisms by which HSV-2, Neisseria gonorrhoeae, and Candida albicans modulate the efficacy of lead microbicides. These experiments will be conducted using human tissue explants from the cervicovaginal mucosa, and CD4+T cells isolated from lower female reproductive tract (FRT) tissues, infected ex vivo with primary isolates of HIV-1. Tissue explants truthfully reflect important aspects of cell-cell and cell-pathogen interactions that occu in the context of the complex tissue cytoarchitecture. Deciphering the mechanisms of these interactions is critical for understanding pathogenic processes caused by human pathogens such as HIV-1. Furthermore, tissue explants are a relevant experimental model for the preclinical evaluation of microbicide candidates that our group and leaders in the field have developed as a surrogate of microbicide efficacy. TLR play a significant role in activating immune responses triggered by STP with prevalence rates ranging from 40 to 80% in young sexually active adults living with HIV-1. Understanding the mechanisms by which TLR activation modulates the anti-HIV-1 activity of lead microbicides is relevant to developing adjunctive therapies that could enhance the efficacy of compounds aimed at preventing mucosal transmission of HIV-1. Furthermore, as the microenvironment of the FRT influences the risk of transmission in the absence of microbicides, our findings will lead to the development of approaches other than microbicides to prevent HIV-1 transmission to women under circumstances of co-infection by STP where they may be the most vulnerable.